Estrogen receptor positive (ER+) luminal breast cancers have a good prognosis, whereas ER- basal breast cancers are highly aggressive, and have poor disease free survival. We recently discovered that ER+ breast tumors contain a subpopulation of progestin (P)-regulated, ER-, basal-like cells. Hypothesis and Objective. We hypothesize that in luminal tumors, P expands a subpopulation of basal-like cancer stem cells (CSCs) leading to estrogen-driven expansion and progression of ER+, PR+ luminal-type breast cancers. Our objective is to determine whether basal-like, ER-/PR-/cytokeratin 5+(CK5+) breast cancer cells are P-regulated, tumorigenic CSCs that give rise to non-tumorigenic, ER+/PR+/CK5- progeny. Research Aims and Design. Specific aim 1 will develop a system to isolate live ER-/PR-/CK5+ cells and then evaluate their CSC properties using live cell imaging, colony-initiation, and tumorigenicity assays. This is accomplished by placing the CK5 promoter upstream of the ZsGreen (ZsG) fluor to identify live CK5+ cells that can be isolated by FACS. Specific aim 2 will determine whether the pool of CK5+ cells expanded by P also have CSC properties and generate luminal tumors. This will be accomplished by isolating CK5+ cells from E and E+P treated luminal breast cancer cells, and measuring their clonogenic (in vitro) and tumorigenic (in vivo) ability. Specific aim 3 will define P-dependent pathways that cause expansion of the CK5+ subpopulation and lead to suppression of ER and PR expression. This will be accomplished by using reporter plasmids containing the human CK5 promoter linked to luciferase, transfecting luminal breast cancer cell lines treated with vehicle or P, and testing inhibitors to several common signaling pathways. PUBLIC HEALTH RELEVANCE: The 70 to 80% of breast cancer patients with ER+ disease could benefit immensely from these studies because: 1) they explain why ER+ breast cancers so often become tamoxifen resistant and relapse, 2) they demonstrate that logical therapies should involve drugs that target ER- cells (the true root of the problem) not just the bulk ER+ tumor cells. These studies also provide a plausible explanation for why women taking progestins in hormone replacement therapy (HRT) were at a higher risk of getting breast cancer. This information could be a guide for doctor's prescribing progestins in other clinical applications.